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Allicin and methicillin-resistant Staphylococcus aureus
Antibacterial activity of a new, stable, aqueousextract of allicin against methicillin-resistantStaphylococcus aureus R R. CUTLER* and P. WILSON† *University of East London, School of Health and Bioscience, Stratford Campus,Romford Road, London E15 4LZ; and Department of Medical Microbiology, The increasing prevalence of methicillin-resistant St Bartholomew's Hospital, West Smithfield, London EC1A 7BE, UK Staphylococcus aureus (MRSA) in hospitals and thecommunity has led to a demand for new agents that couldbe used to decrease the spread of these bacteria. Topical Accepted: 19 March 2004 agents such as mupirocin have been used to reduce nasalcarriage and spread and to treat skin infections; however,resistance to mupirocin in MRSAs is increasing. Allicin is the main antibacterial agent isolated from garlic, butnatural extracts can be unstable. In this study, a new, stable, Control of the spread of antibiotic-resistant bacteria and the aqueous extract of allicin (extracted from garlic) is tested on treatment of infections caused by them is a major problem 30 clinical isolates of MRSA that show a range of worldwide. In particular, methicillin-resistant Staphylococcus susceptibilities to mupirocin. Strains were tested using agar aureus (MRSA) presents major infection control problems for diffusion tests, minimum inhibitory concentration (MIC) patients and hospital staff, as its incidence in Europe has and minimum bactericidal concentration (MBC). Diffusion risen from 3% in 1992 to 37% in 1999.1–4 tests showed that allicin liquids produced zone diameters Topical agents are important in controlling the carriage >33 mm when the proposed therapeutic concentration of and spread of MRSA. Mupirocin (pseudomonic acid), a 500 µg/mL (0.0005% w/v) was used. The selection of this fermentation product produced by Pseudomonas fluorescens concentration was based on evidence from the MIC, MBC (NCIB 10586), is a standard product used to deal with MRSA and agar diffusion tests in this study. Of the strains tested, carriage and to prevent its spread. It has also proved to be an 88% had MICs for allicin liquids of 16 µg/mL, and all strains effective treatment for skin infections and plays a crucial role were inhibited at 32 µg/mL. Furthermore, 88% of clinical in the control of MRSA outbreaks. isolates had MBCs of 128 µg/mL, and all were killed at 256 However, resistant strains were described soon after its µg/mL. Of these strains, 82% showed intermediate or full introduction. Moreover, the increased use of mupirocin, resistance to mupirocin; however, this study showed that a especially for chronic infections, has led to an increased concentration of 500 µg/mL in an aqueous cream base was incidence of resistance. In a recent survey from Spain, levels required to produce an activity equivalent to 256 µg/mL of mupirocin resistance in clinical isolates was reported to have increased from 7.7% in 1998 to 19% in 2000, and somehospitals have reported incidences as high as 63%.2 The continuing spread of MRSA and the increase in Drug resistance, microbial. mupirocin-resistant strains5 highlight the need for alternative topical agents Methicillin resistance. Garlic and its extracts have been used to treat infections Staphylococcus aureus. for thousands of years.6 Allicin (the name being derived from Sulfinic acids.
that of the garlic species Allium sativum) is considered to bethe main biologically active antimicrobial phytochemicalproduced in garlic extracts, and was first recognised as such Pure allicin (allyl 2-propenethiosulphinate)6 is said to be highly volatile, poorly miscible with water and has the Allicin is an oxygenated sulphur compound, formed when odour of freshly crushed garlic.9 In order to produce a stable garlic cloves are crushed. Alliin is the stable precursor of agent that can be used in topical formulations, an aqueous allicin and is stored in compartments in the plant that allicin extract is needed. separate it from the enzyme alliinase (also called alliin lyase).
In this study, a purified aqueous extract of allicin, isolated When crushed, they mix and alliin is converted rapidly to from a natural source by a patented cold aqueous extraction allicin by the action of this enzyme. The antibacterial activity method (Allicin International, Half House, Military Road, of allicin was reviewed by Ankri and Mirelman in 1999.8 Rye, East Sussex, UK) is used. This extract is tested againstmupirocin-resistant and mupirocin-susceptible strains of Correspondence to: Dr Ron Cutler MRSA. Two formulations, liquid allicin and liquid allicin mixed in a cream formulation, are tested. BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2004 61 (2)

Allicin and methicillin-resistant Staphylococcus aureus
Number of strains Fig. 1. Number of strains and the size of zones of inhibition withallicin (500 µg/mL) and mupirocin against Staphylococcus aureus.
Materials and methods Concentration of allicin (ug/ml) Bacterial strains Fig. 2. Average diameter of zones of inhibition for allicin liquid Thirty clinical isolates and one control strain (Oxford Strain, against Staphylococcus aureus strains that showed resistance, NCTC 6571) of S. aureus were tested. All 30 clinical isolates intermediate resistance or sensitivity to mupirocin. were obtained from the Royal London and St. Bartholomew'sHospitals and had been identified as showing multipleantibiotic resistance. All strains were resistant to methicillin The bacterial inoculum used was approximately Log 6 and had no apparent epidemiological connection colony-forming units (cfu)/mL. Cultures were incubatedovernight at 37˚C and examined for growth the following day. Subcultures of these were used to determine MBC.12 A 5000 µg/mL solution of allicin in water was provided byAllicin International. As it was an extracted product, thepurity of the allicin solution was tested and the concentration confirmed by Allicin International using thehigh-performance liquid chromatography (HPLC) method of Lawson, Wang and Hughes.10 The control strain produced a 35-mm diameter zone ofinhibition to mupirocin. Of the clinical isolates, five strains were identified as fully susceptible (zone sizes 33–45 mm), 12 The allicin liquid and allicin made up in aqueous cream were showed intermediate susceptibility (zone sizes 12–23 mm) tested for antimicrobial activity against MRSA using an agar and 13 strains were resistant (no zone of inhibition).
well diffusion method. Muller-Hinton agar plates were Variability of zone size was considerable, ranging from 0–45 inoculated using the methods recommended by the British mm. The most frequent result (13 strains) showed no zone of Society for Antimicrobial Chemotherapy (BSAC).11 inhibition. The second most common group (11 strains) Circular wells (6 mm) were cut in the agar culture media produced intermediate zone sizes (8–19 mm). Two strains and filled with 100 µL cream or liquid. The accuracy of the were highly susceptible, producing zone sizes of 45 mm.
method used to deliver these volumes was confirmed by weighing the agar plates before and after the fluid andcream were added.
Allicin liquid activity Mupirocin activity against the same MRSA strains was Allicin liquid was active against S. aureus strains down to 62.5 determined using a 5 µg paper disk (Oxoid, UK) on Muller- µg/mL (equal to 6.25 µg of allicin in 100 µL [the volume Hinton agar plates added to each well]). No activity was detected below 62.5 µg/mL, and concentrations of 250 µg/mL and above were the Minimum inhibitory and bactericidal concentrations of allicin most active.
The average zone sizes produced against these strains Standard methods based on those of Lorian12 were used to were 25 mm at 250 µg/mL, 32 mm at 500 µg/mL and 38 mm determine minimum inhibitory concentration (MIC) and at 1000 µg/mL. The spread of zone size to allicin was smaller minimum bactericidal concentration (MBC). Strains were than that found with mupirocin (Fig. 1). Zone sizes ranged cultured overnight at 37˚C in Muller-Hinton broth (Oxoid, from 23–38 mm at 500 µg/mL, and from 31–44 mm at 1000 CM 405). Concentrations of allicin between 1–1000 µg/mL µg/mL (Figs. 2 and 3). and broth containing no antimicrobial agent (negativecontrol) were assessed. Creams were not tested by this Allicin cream activity method because the opacity they produced in the liquid The control strain produced a 20 (±1.5) mm zone of inhibition made determination of MIC impossible.
compared to 23 (± 1.5) mm for mupirocin-susceptible MRSAs BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2004 61 (2)

Allicin and methicillin-resistant Staphylococcus aureus
Concentration of allicin (ug/ml) Fig. 3. A typical zone of inhibition produced by 500 µg/mL allicin Fig. 4. Average diameter of zones of inhibition for allicin cream against MRSA.
against Staphylococcus aureus strains that showed resistance,intermediate resistance or sensitivity to mupirocin.
and 21 (±1.5) mm for mupirocin-resistant MRSAs. When Table 1. Susceptibility of MRSA clinical isolates to a stabilised allicin prepared in Boots' aqueous cream, allicin was found to be extract, shown as the percentage of strains with different minimal highly active against all strains at a concentration of 500 inhibitory concentrations (MIC) and minimum bactericidal µg/mL. No activity was detected below 125µg/mL (Fig. 4).
Comparison of allicin activity in cream and liquidThe average zone size for 250 µg/mL allicin liquid was 25 (±1) mm. This was almost identical to the average zone size of 23.8 (±2) mm obtained with 500 µg/mL allicin in aqueous cream.
Minimum inhibitory and bactericidal concentrations The control strain tested (Oxford S. aureus) gave an MIC of32 µg/mL and an MBC of 256 µg/mL. The MICs for theclinical isolates tested were either 16 or 32 µg/mL, the MBCs Allicin is considered to be the most potent antibacterial were either 128 or 256 µg/mL. The majority of the clinical agent in crushed garlic extracts, but it can be unstable, isolates had MICs of 16 µg/mL and MBCs of 128 µg/mL breaking down within 16 h at 23˚C.6 However, the use of a water-based extract of allicin stabilises the allicin molecule.
Of the clinical isolates, 88% had MICs of 16 µg/mL and This may be due to two factors: the hydrogen bonding of MBCs of 128 µg/mL, 18% were susceptible to mupirocin (as water to the reactive oxygen atom in allicin can reduce its indicated by the disk diffusion test), 47% showed instability; and/or there may be water-soluble components in intermediate susceptibility and 35% were resistant. All crushed garlic that destabilise the molecule.14 mupirocin-resistant strains had MICs of 16 µg/mL, 23% had The disadvantage to this approach is that allicin can react MBCs of 128 µg/mL and 12% had MBCs of 256 µg/mL. with water to form diallyl disulphide,15,16 which does notexhibit the same level of antibacterial activity as does allicin.
In 1991, Hughes and Lawson17 reported tests on a single strain of S. aureus and noted that pure allicin had an MIC of27 µg/mL compared with 900 µg/mL for diallyl disulphide. In a review of the subject, Schmitz and Jones13 considered In the present study, 88% of strains had MICs for allicin of the choices available in the battle against MRSA and 16 µg/mL, and all strains were inhibited by allicin at 32 concluded that only vancomycin as a systemic agent and µg/mL. This compares well with Lawson's results with mupirocin as a topical agent offered reliable treatment. They purified allicin on a single isolate. also pointed out that reports of mupirocin resistance had There is always concern that antibiotic resistance to new increased since 1990. The emergence of epidemic MRSA agents may develop rapidly when they have similar modes resistant to mupirocin has led many authors to suggest that of action to established agents, and can be a particular the use of mupirocin should be controlled more strictly, problem with topical agents. This, however, is not the case especially as there is a lack of alternative agents.
with allicin and mupirocin. The S(=O)S thiosulphinate Consequently, alternative agents for topical use against group in allicin is thought to react with a variety of SH- MRSA would prove very popular and be in great demand containing enzymes within the bacterial cell, and allicin has BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2004 61 (2)
Allicin and methicillin-resistant Staphylococcus aureus
been reported to have a range of potential targets. It is Schentag JJ, Hyatt M, Carr J et al. Genesis of methicillin-resistant reported to inhibit the acetyl CoA forming system, to inhibit Staphylococcus aureus (MRSA), how treatment of MRSA DNA and protein synthesis, and to target RNA infections has selected for vancomycin-resistent Enterococcus polymerase;18–20 and these are responsible for the agents faecium and the importance of antibiotic management and antibacterial effect. infection control. Clin Infect Dis 1998; 26: 1204–14.
More general proposals about the broad-spectrum activity Walker ES, Vasquez JE, Dula R, Bullock H, Sarubbi FA.
of allicin were provided by Rabinkov et al. in 1998. This Mupirocin-resistant, methicillin-resistant Staphylococcus aureus: group compared the importance of its antioxidant properties does mupirocin remain effective? Infect Control Hosp Epidemiol with its thiol disulphide exchange activity and suggested 2003; 24: 342–6.
that activity is related to allicin's rapid reaction with thiol- Hahn G. In: Koch HP, Lawson LD, eds. Garlic: the science and containing proteins. In contrast, mupirocin (pseudomonic therapeutic application of Allium sativum L and related species (2nd acid) inhibits protein synthesis by slowing the activity of edn). Baltimore Williams and Wilkins, 1996: 1–24.
RNA synthetase.13 Cavallito C, Bailey JH. Allicin, the antibacterial principal of For a topical agent to produce maximum benefit, its Allium sativum. Isolation, physical properties and antibacterial strength in any formulation should not only be sufficient to action. J Am Chem Soc 1944; 66: 1944–52.
inhibit growth but also to be bactericidal. The present study Ankri S, Mirelman D. Antimicrobial properties of allicin from demonstrated that the majority (88%) of strains had MBCs garlic. Microbes Infect 1999; 2: 125–9.
for allicin of 128 µg/mL and all the strains were killed by Block E. The chemistry of garlic and onion. Sci Am 1985; 252:
allicin at 256 µg/mL. Allicin liquid extracts were highly active against clinical 10 Lawson LD, Wang ZJ, Hughes BG. Identification and HPLC of isolates of multiple antibiotic resistant S. aureus, including the sulphides and dialk(en)yl thiosulphinates in commercial those strains that were identified as mupirocin-resistant.
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Although agar diffusion tests showed that the activity of 11 Andrews JM. BSAC standardised disc susceptibility testing allicin was reduced in the cream formulation, comparison of method. J Antimicrob Chemother 2001; 48 (Suppl S1): 5–16.
zone sizes achieved with 250 µg/mL in liquid showed 12 Lorian V. Antibiotics in laboratory medicine. Baltimore: Williams similarity to those achieved with 500 µg/mL in cream and Wilkins, 1980.
(average zone sizes of 25 mm and 23.7 mm, respectively). 13 Schmitz FJ, Jones ME. Antibiotics for treatment of infections When made up in an aqueous cream base, 500 µg/mL caused by MRSA and elimination of MRSA carriage. What are allicin produced bactericidal levels high enough to support the choices? Int J Antimicrob Agents 1997; 9: 1–19.
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processed garlic. In: Koch HP, Lawson LD, eds. Garlic: the science In conclusion, the present study demonstrated that liquid and therapeutic application of Allium sativum L and related species and cream formulations containing allicin are active against (2nd edn). Baltimore: Williams and Wilkins, 1996: 37–107.
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18 Feldberg RS, Chang SC, Kotic AN et al. In vitro mechanism of inhibition of bacterial cell growth by allicin. Antimicrob Agents Chemother 1988; 32: 1763–8.
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Perez-Roth E, Claverie-Martin F, Batista N, Moreno A, Mendez- Specific modification of the alpha-subunit of Escherichia coli Alvarez S. Mupirocin resistance in methicillin-resistant RNAS polymerase by monomercuric derivative of flourescein Staphylococcus aureus clinical isolates in a Spanish hospital. Co- mercuric acetate. Mol Biol (Mosk) 1990; 24: 1057–66.
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